Background: New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important. Results: Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution. Conclusions: Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.

The kombucha market is a fast-growing segment in the functional beverage category. The selection of kombuchas on the market varies between the traditional and flavoured kombuchas. Our research aimed to characterise the chemical, microbial, and sensory profiles of the commercial kombuchas. We analysed 16 kombuchas from 6 producers. The dominant metabolites were acetate, lactate, and ethanol, the last of which might put some kombuchas into the alcoholic beverage section in some countries. The metagenomic analyses demonstrated that LAB dominates in green tea, and AAB in black tea kombuchas. The main bacterial species were Komagataeibacter rhaeticus and Lactobacillus ssp, and yeast species Dekkera anomala and Dekkera bruxellensis. The sweet and sour balance correlated with acid concentrations. The free sorting task showed that commercial kombuchas clustered into three main categories "fruity and artificial flavour", herbal and tea notes", and "classical notes". Our research results showed the necessity of the definition of kombucha.

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox170S–S binds co-operatively to four Cu+; Cox172S–S, with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox173S–S, with three disulfide bridges, does not bind to any metal ions. The Em (midpoint redox potential) values for two redox couples of Cox17, Cox173S–S↔Cox172S–S (Em1) and Cox172S–S↔Cox170S–S (Em2), were determined to be −197 mV and −340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox170S-S and Cox172S–S, which is slightly shifted towards Cox170S-S. In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox172S–S, enabling retention of Cox172S–S in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox172S–S, capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper–thiolate cluster, which may provide safe storage of an excess of copper ions.

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota.